cAMP-dependent protein kinase (PKA), ubiquitous in mammalian cells, regulates a plethora of cell processes including development, differentiation, memory, and metabolism and is associated with many diseases including multiple endocrine disorders and cancer. It serves as a prototype for the protein kinase superfamily and its activation by cAMP is a classic example of allosteric regulation. While our studies of PKA structure and function have given us a molecular understanding of individual PKA catalytic and regulatory subunits, over the past four years we have added a new dimension to our understanding of PKA signaling by solving structures of PKA tetrameric holoenzymes. These first glimpses of the full-length proteins force us to appreciate that PKA functions as a complex and multicomponent signaling system. Protein kinases have evolved to be dynamic and highly regulated molecular switches, not efficient catalysts, and we simply cannot appreciate or understand how this PKA signaling system works and how it is allosterically regulated by cAMP without seeing the full-length holoenzymes. It is these large multi-domain tetrameric complexes that represent the physiological state of PKA signaling in cells, and the quaternary structure of each isoform (RI, RI, RII, and RII) is different, as we had predicted based on our low resolution SAXS analyses. Our goals now are to further define the molecular features of these holoenzymes as integrated and finely tuned systems using RI and RII holoenzymes as prototypical isoforms that nucleate dynamic multicomponent macromolecular PKA signaling complexes. Aim I focuses on RI and uses a disease mutation of RI to dissect the pathway for allosteric signaling from one cAMP-binding domain to the other. Crystal structures of the full-length mutant holoenzyme will be validated using solution methods such as SAXS and SANS while mutagenesis will confirm interfaces and provide functional, mechanistic insights. To build higher levels of RI complexity we focus on two newly discovered proteins. P-Rex1, up-regulated in cancers, is a guanine nucleotide exchange factor for Rac1 that binds through its two PDZ domains to the C-terminal PDZ motif of RI. To the N-terminal D/D domain we are adding a small newly discovered membrane associated RI-specific AKAP, smAKAP. In Aim II we are building a quantitative model for PKA signaling by RII. We specifically define the role of phosphatases, metals (both Mg2+ and Ca2+), and phosphodiesterases. We introduce new concepts by showing the importance of the turnover of a single phosphate in RII and emphasize that the release of cAMP from RII is not diffusion limited. Finally, in Aim III we build higher levels of complexity for the RII signalosome by adding a dual-specific AKAP, DAKAP2, to the holoenzyme. DAKAP2 is a multi-domain AKAP that regulates recycling by binding through its PDZ motif to PDZ-K1, which links it to transporters, and through its RGS domains that bind to Rabs. In parallel with crystallography, we will use small angle Xray and neutron scattering as well as single particle image reconstruction to monitor conformational changes and domain organization in solution.